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Method of freezing cells

a cell culture and cell technology, applied in the field of multi-layer cell freezing vials, can solve the problems of microbial contamination, laborious and time-consuming cell culture methods, and low cell number, and achieve the effect of reducing the number of cells

Inactive Publication Date: 2014-01-02
ZF BIOTOX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention is a method for freezing and thawing cells without needing to add new fresh medium to dilute the cryoprotective agent. This is achieved by previously freezing an extra layer of medium or diluent that contains the cryoprotective agent. When the cells are thawed, the diluent and cell solution mix, resulting in a non-pathogenic dilution of the cryoprotective agent. This separation of the cryoprotective agent from the living cells reduces cell expansion and saves time during subsequent assays. The method can be used with either a higher or lower molecular weight cryoprotective agent than the diluent.

Problems solved by technology

Until the middle 80's, cell culture techniques were labor-intensive and did not scale to high cell numbers.
This tissue culture process is still the current standard for seeding cells in a high throughput format, being a laborious and time consuming method that requires proper cell culture facilities and qualified technicians.
The result is exposed to microbial contamination and cross-contamination of the cells due to the numerous handling procedures involved.
However, it is well known that cells frozen in monolayers are more susceptible to freezing injury than those in suspension.
The problem of the art is then to provide a freezing method that could result in better post-thawing cell viability, in the save of time at tests performance and in diminish contamination risks.

Method used

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Examples

Experimental program
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Effect test

example 1

Two-Layer Cell Freezing / Thawing Method on HT29 Colon Carcinoma Cells

[0054]HT29 Cells (ATCC, LGC Standards) cultured in endotoxin-free McCoy's 5A complete culture medium supplemented with 10% FCS (Gibco), 100 μg / ml penicillin and 100 IU / ml streptomycin (Sigma-Aldrich) were grown in flasks until they were 80% confluent, and than harvested. Harvesting took place by incubating cells with 5 ml of PBS containing 0.05% EDTA for 2 min, and final detachment was achieved by incubation in 5 ml of PBS with 0.1% trypsin and 0.05% EDTA for another 5 min. Cells were centrifuged at 400×G for 5 min. Then, a total of 4.8 ml of a solution at 4° C. containing 80,000 HT29 cells / 50 μl of freezing 80% Mc Coy's, 10% FCS, 10% DMSO (AppliChem) medium was prepared (cell density determined by haemocytometer), forming the no called freezing solution. Besides, 100 μl of complete culture medium was disposed in each cell of a 96-well plate and frozen down to −80° C. Once frozen, the plate is recovered and 50 μl / we...

example 2

Two-Layer Cell Freezing / Thawing Method on NIH3T3 Murine Fibroblasts

[0057]NIH3T3 cells (ATCC, LGC Standards) cultured in endotoxi-free DMEM medium (Sigma-Aldrich) supplemented with 10% FCS, 100 μg / ml penicillin and 100 IU / ml streptomycin (Sigma-Aldrich) were grown in flasks until they were 80% confluent, and then harvested. Harvesting took place by incubating cells in 5 ml of PBS with 0.05% EDTA for 2 minutes, and final detachment was achieved by incubation in 0.1% trypsin and 0.05% EDTA for another 5 minutes. Cells were centrifuged at 400×G for 5 minutes. Then a total of 4.8 ml of a solution at 4° C. containing 80,000 NIH3T3 cells / 50 μl of freezing 80% DMEM, 10% FCS, 10% DMSO (AppliChem) medium was prepared (cell density determined by haernocytometer), forming the so-called freezing solution. Besides, 100 μl of complete culture medium was disposed in each cell of a 96-well plate and frozen down to −80° C. Once frozen, the plate is recovered and 50 μl / well of the said freezing soluti...

example 3

Cytotoxic Test

[0060]Balb3T3 (ATCC, LCC Standards) fibroblasts and smooth muscle aortic cells (SMAC, freshly isolated from Sprague Dawley rats, Harlan Laboratories) primary cells were cultured in complete culture medium consisting of endotoxin-free DMEM (Sigma-Aldrich) supplemented with 10% FCS, 100 μ / ml penicillin and 100 IU / ml streptomycin (Sigma-Aldrich). SMAC cells were isolated from a single male rat aorta: the aorta was carefully dissected from its origin at the left ventricle to the iliac bifurcation and cells were isolated by enzymatic digestion of the tissue with collagenase type II (Worthington). The resulting cells were allowed to grow for twelve days prior to cryopresentation. Both cell types were grown in flasks until they were 80% confluent and then harvested. Harvesting took place by incubating cells in 5 ml of PBS with 0.05% EDTA for 2 minutes, and final detachment was achieved by incubation in 0.1% trypsin and 0.05% EDTA for another 5 minutes. Cells were centrifuged ...

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PUM

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Abstract

The invention discloses a multi-layer cell freezing vial and method of ohtention thereof. The method of obtention is a cell freezing and thawing process that avoids the need of incorporating new fresh medium to the thawed cells for the dilution of the cryoprotective agent. This is achieved by the previous freezing of an extra layer of culture medium in addition to the frozen ceil solution containing said cryoprotective agent. At the time of thawing, the culture medium and the cell solution mix themselves, turning the concentration of said cryoprotective agent to a non-toxic dilution and achieving the effective exclusion of said cryoprotective agent from the living cells.

Description

FIELD OF THE INVENTION[0001]The invention discloses a multi-layer cell freezing vial and method of obtention thereof. The viability and durability of the frozen cells is increased after the method of the Invention. It is useful in cell-based assays for basic research, diagnostics and drug development applications in the biotechnology and pharmaceutical sector.BACKGROUND ART[0002]In vitro cultured cells, including mammalian cells, are extensively used in cell biology studies. Until the middle 80's, cell culture techniques were labor-intensive and did not scale to high cell numbers. Currently, there is a wide acceptance and universal application of High Throughput Screening (HTS) using cell-based assays in the Pharmaceutical and Biotechnology industry as well as in basic research.[0003]HTS screening is performed in microplates. The surface of these microplates may be modified for cell culturing applications, typically using a plasma discharge for easier cell attachment. These cell cul...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/0284A01N1/0221A01N1/0268
Inventor HERNAN IZQUIERDO, ROBERTOGALLOT ESCOBAL, NATALIACRUZ PACHECO, ANTONIO
Owner ZF BIOTOX
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